Hepatocellular carcinoma (HCC) represents an extremely poor prognostic cancer that remains one of the most common and aggressive human malignancies worldwide (1; 2). The dismal outcome has been attributed to the major hallmarks of HCC, intra-hepatic metastases or post-surgical recurrence. New tumor colonies frequently invade into the major branches of the portal vein and possibly other parts of the liver (3-6). Resection or liver transplantation, are the best options for a potential cure however, only about 20 percent of HCC patients, defined by parameters of relatively normal liver function and a manageable tumor lesion as determined by the available clinical staging systems, are currently eligible for surgical intervention. Moreover, resected patients often have a high frequency of metastasis/recurrence, and post-operative 5 year survival is only 30-40 percent.
Liver transplantation for HCC patients remains controversial due to a shortage of organ donors and the poor performance of current staging systems in selecting appropriate candidates, especially at early disease stages. These systems are essential, particularly in malignant diseases, to provide advice to patients and guidance for assessment and treatment. Clinical evaluation and therapeutic decisions in HCC is complex because they depend on both the grade of cancer spread (tumor staging) and residual liver function (chronic liver disease stage). Although well-defined and generally accepted staging systems are available for almost all cancers, HCC is an exception, with many different staging systems globally introduced to accommodate each stratum of the disease and a current lack of consensus on which one is best (7-12). Thus, an accurate prognostic predictor and a sensible selection criterion that can be applied to HCC patients for rational treatment decisions remains a challenging task.
The recent identification of prognostic molecular biomarkers offers hope for advance diagnosis of HCC. Using cDNA microarray technology, the inventors developed a unique gene expression signature to predict prognosis and metastasis of HCC patients (13). The presence of a molecular prognostic signature in primary HCC clinical specimens was confirmed by several recent studies (14; 15). Since HCC is usually present in inflamed liver, the inventors also developed a unique predictor based on the expression of genes in the liver microenvironment of HCC patients, which was principally different from that of the tumor (16). Like many other prognostic signatures based on cDNA gene expression profiling, both the tumor and microenvironment signatures contain several hundred cellular coding genes. Therefore, it would be a challenging task to identify relevant biomarkers or potential pharmacological targets and interrogate scores of genes in clinical practice.
Recent studies indicate that expression profiling with small non-coding RNA gene products (−22 nt) known as microRNAs (miRNAs or miRs) is a superior method for cancer subtype classification and prognostication (17-19). miRNAs exist in many organisms and play key regulatory roles in mRNA translation and degradation by base pairing to partially complementary sites of the mRNA, predominantly in the 3′ untranslated region (20-22). miRNAs are expressed as long precursor RNAs that are processed by Drosha, a cellular nuclease, and subsequently transported to the cytoplasm by an Exportin-5-dependent mechanism (23; 24). miRNAs are then cleaved by the DICER enzyme, resulting in—17-24 nt miRNAs that associate with a RNA-induced silencing-like complex (25; 26). The expression patterns, function and regulation of miRNAs in normal and neoplastic human cells are largely unknown but emerging data and their frequent location at fragile sites, common break-points or regions of amplification or loss of heterozygosity reveal that they may play significant roles in human carcinogenesis.
The enhanced expression of precursor miR-155 in Burkitt's lymphomas and the frequent deletion or downregulation of several miRNAs have been observed in B cell chronic lymphocytic leukemia (CLL) and in many cancer types, including breast, lung, ovarian, cervical, colorectal, prostate, and lymphoid (17; 18; 27-34). Functional analysis has also revealed the downregulation of PTEN by miR-21, the tumor suppressor function of the let-7 family and the oncogenic function of the miR1 7-92 cluster (35-37). The biological and clinical relevance of miRNA expression patterns have been shown in human B cell CLL and solid tumors, including breast cancers (18; 30; 38). Each miRNA has the unique capability to potentially regulate the expression of hundreds of coding genes and thereby modulate several cellular pathways including proliferation, apoptosis and stress response (39). This phenomenon makes miRNAs superior molecular markers and targets for interrogation and as such, miRNA expression profiling can be utilized as a tool for cancer diagnosis (17; 40).